EP3245287A1 - Purification de cellules souches germinales par ciblage de mrp9 - Google Patents

Purification de cellules souches germinales par ciblage de mrp9

Info

Publication number
EP3245287A1
EP3245287A1 EP16737697.9A EP16737697A EP3245287A1 EP 3245287 A1 EP3245287 A1 EP 3245287A1 EP 16737697 A EP16737697 A EP 16737697A EP 3245287 A1 EP3245287 A1 EP 3245287A1
Authority
EP
European Patent Office
Prior art keywords
mrp9
antibody
sample
gsc
antibodies
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP16737697.9A
Other languages
German (de)
English (en)
Other versions
EP3245287A4 (fr
Inventor
Michael B. SCHULTZ
Michael S. BONKOWSKI
David A. Sinclair
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Harvard College
Original Assignee
Harvard College
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Harvard College filed Critical Harvard College
Publication of EP3245287A1 publication Critical patent/EP3245287A1/fr
Publication of EP3245287A4 publication Critical patent/EP3245287A4/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0608—Germ cells
    • C12N5/0609—Oocytes, oogonia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1456—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
    • G01N15/1459—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals the analysis being performed on a sample stream
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/149—Optical investigation techniques, e.g. flow cytometry specially adapted for sorting particles, e.g. by their size or optical properties
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01R—MEASURING ELECTRIC VARIABLES; MEASURING MAGNETIC VARIABLES
    • G01R33/00—Arrangements or instruments for measuring magnetic variables
    • G01R33/12—Measuring magnetic properties of articles or specimens of solids or fluids
    • G01R33/1276—Measuring magnetic properties of articles or specimens of solids or fluids of magnetic particles, e.g. imaging of magnetic nanoparticles

Definitions

  • OSCs oogonial stem cells
  • DDX4 protein DEAD box polypeptide 4
  • GSCs germ stem cells
  • OSCs oogonial stem cells
  • SSCs spermatogonial stem cell
  • a method for purifying a GSC based on its expression of Multidrug Resistance- Associated Protein 9 comprises contacting a sample comprising the GSC with an anti-MRP9 antibody that specifically binds to MRP9.
  • the anti-MRP9 antibody does not specifically bind to DDX4.
  • the method comprises incubating the sample under conditions such that the anti-MRP9 antibody forms a complex with an MRP9 protein expressed on the surface of the GSC.
  • the method comprises the step of separating the GSC from other material present in the sample.
  • the GSC is separated from other material present in the sample by fluorescent activated cell sorting (FACS) (e.g., in embodiments wherein the anti-MRP9 antibody is linked to a fluorescent label or contacted with a secondary antibody that is linked to a fluorescent label).
  • FACS fluorescent activated cell sorting
  • MCS magnetic activated cell sorting
  • the GSC is separated from other material present in the sample by adhesion-based cell sorting (e.g., in embodiments wherein the anti- MRP9 antibody is immobilized on a solid support or contacted with a secondary antibody that is immobilized on a solid support).
  • the method includes contacting the sample with one or more additional GSC-specific antibodies and incubating the sample under conditions such that the additional anti-GSC specific antibodise form a complex with their target proteinon the surface of the GSC.
  • the additional GSC-specific antibody is an anti- DDX4 antibody.
  • the additional GSC-specific antibody is an anti- Fragilis antibody.
  • the additional GSC-specific antibodies include both an anti-DDX4 antibody and an anti-Fragilis antibody.
  • provided herein is a method for generating a culture of GSCs.
  • the method comprises purifying a GSC based on its expression of MRP9 according to the methods described herein and then culturing the GSC.
  • provided herein is a method for generating GSC
  • mitochondria e.g., OSC mitochondria
  • the method comprising purifying a GSC based on its expression of MRP9 according to the methods described herein and then isolating the mitochondria from the purified GSCs.
  • the isolated mitochondria are used in an autologous germline mitochondrial transfer-IVF (AUGMENT-IVF) procedure, for example, as described in Tilly and Sinclair Cell Metabolism 17:838-850 (2013) which is hereby incorporated by reference in its entirety.
  • Exemplary methods for isolating mitochondria and transferring such mitochondria into oocytes for the performance of a AUGMENT-IVF procedure are provided in, for example, U.S. Pat. No. 8,642,329, which is hereby incorporated by reference in its entirety.
  • the method includes the step of contacting a sample comprising the GSC with an anti-MRP9 antibody that specifically binds to MRP9. In some embodiments, the antibody does not specifically bind to DDX4. In some embodiments, the method includes the step of incubating the sample under conditions such that the anti-MRP9 antibody forms a complex with an MRP9 protein expressed on the surface of the GSC. In some embodiments, the method includes the step of detecting the GSC by detecting the complex. In some embodiments, the GSC is detected by fluorescence microscopy or FACS. In some embodiments, the method further comprises purifying the detected GSC from other material present in the sample.
  • the GSC is a OSC.
  • the sample is an ovarian tissue sample.
  • the method further comprises the step of obtaining the ovarian tissue sample from a subject (e.g., a human subject).
  • the GSC is a SSC.
  • the GSC is a mammalian GSC.
  • the GSC is a human GSC.
  • the anti-MRP9 antibody is monoclonal. In some embodiments, the anti-MRP9 antibody is polyclonal. In some embodiments, the anti-MRP9 antibody specifically binds to an extracellular region of MRP9. In some embodiments, the anti-MRP9 antibody does not specifically bind to an epitope of MRP9 having a sequence of AP PVDD. In some embodiments, the anti-MRP9 antibody specifically binds to an extracellular region of MRP9 having sequence selected from the group consisting of SEQ ID NOs 5-20. In some embodiments, the anti-MRP9 antibody specifically bind to an extracellular region of MRP9 having sequence selected from the group consisting of SEQ ID NOs 6-12.
  • the anti-MRP9 antibody, the one or more additional anti-GSC antibodies and/or the secondary antibody is linked to a detectable label (e.g., a fluorescent moiety, a radioactive moiety, a paramagnetic moiety, a luminescent moiety and/or a colorimetric moiety).
  • a detectable label e.g., a fluorescent moiety, a radioactive moiety, a paramagnetic moiety, a luminescent moiety and/or a colorimetric moiety.
  • the anti-MRP9 antibody, the one or more additional anti-GSC antibodies and/or the secondary antibody is immobilized on a solid support.
  • the method further comprises the steps of contacting the sample with a secondary antibody linked to a detectable label that specifically binds to the anti-MRP9 antibody and incubating the sample under conditions such that the secondary antibody forms a complex with the anti- MRP9 antibody. In some embodiments of the methods described herein, the method further comprises the steps of contacting the sample with a secondary antibody linked to a solid support that specifically binds to the anti-MRP9 antibody and incubating the sample under conditions such that the secondary antibody forms a complex with the anti-MRP9 antibody.
  • the method further comprises contaicting the sample with a detectable label that distinguishes live cells from dead cells.
  • the detectable label that distinguishes live cells from dead cells is 4', 6-doa,odome-2-phenylindole (DAPI), propidium iodide (PI), 7 Amino- Actinomycin D (7-AAD), TO-PRO-3, and/or a Calcein Dye (e.g., Calcein AM, Calcein Violet AM, Calcein Blue AM).
  • Figure 1 shows that a DDX4 antibody (Abeam 13840) does not bind DDX4 on live cells.
  • Panel (a) depicts a model of antibody binding in live compared to fixed and permeabilized cells.
  • Panel (b) depicts a western blot of DDX4 knockdowns with the DDX4 antibody.
  • Panel (c) depicts the analysis of DDX4 antibody affinity by flow cytometry.
  • Figure 2 shows that a DDX4 antibody (Abeam 13840) binds to MRP9 on live cells.
  • Panel (a) depicts the quantification of DDX4 antibody affinity against OSCs treated with siScrambled, siMRP9 #2, or siMRP9#3, as determined by flow cytometry.
  • Panel (b) is a schematic depiction of the amino acid sequence of MRP9, showing the epitopes of the DDX4 and MRP9 antibodies.
  • Panel (c) depicts a western blot of MRP9 knockdowns with the MRP9 antibody.
  • Figure 3 depicts a human MRP9 mRNA sequence.
  • Figure 4 depicts a human MRP9 amino acid sequence with the putative
  • Figure 5 depicts a mouse MRP9 mRNA sequence.
  • Figure 6 depicts a mouse MRP9 amino acid sequence with the putative
  • Figure 7 shows the results of a western blot demonstrating that DDX4 is not expressed on the cell surface of OSCs, while MRP9 is present in the cytosol and on the membrane of OSCs.
  • GSCs germ stem cells
  • OSCs oogonial stem cells
  • SSCs spermatogonial stem cells
  • MRP9 Multidrug Resistance-Associated Protein 9
  • DDX4 DEAD box polypeptide 4
  • MRP9 unlike DDX4, is expressed on the surface of OSCs and is therefore a better target for the purification and/or detection of live cells.
  • antibodies that specifically bind to MRP9 and that are either directly or indirectly associated with a detectable label can be used to detect and/or purify MRP9 expressing GSCs using a wide range of methodology, including fluorescent activated cell sorting (FACS), magnetic activated cell sorting (MACS), adhesion-based cell sorting and fluorescence microscopy.
  • FACS fluorescent activated cell sorting
  • MCS magnetic activated cell sorting
  • adhesion-based cell sorting adhesion-based cell sorting
  • fluorescence microscopy fluorescence microscopy.
  • antibody may refer to both an intact antibody and an antigen binding fragment thereof.
  • Intact antibodies are glycoproteins that include at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds.
  • Each heavy chain includes a heavy chain variable region (abbreviated herein as V H ) and a heavy chain constant region.
  • Each light chain includes a light chain variable region (abbreviated herein as V L ) and a light chain constant region.
  • the V H and V L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • Each V H and V L is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.
  • the term "antibody” includes, for example, monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies and antigen-binding antibody fragments.
  • antigen binding fragment' and "antigen-binding portion" of an antibody refers to one or more fragments of an antibody that retain the ability to bind to an antigen.
  • binding fragments encompassed within the term "antigen-binding fragment” of an antibody include Fab, Fab', F(ab') 2 , Fv, scFv, disulfide linked Fv, Fd, diabodies, single-chain antibodies, NANOBODIES®, isolated CDRH3, and other antibody fragments that retain at least a portion of the variable region of an intact antibody. These antibody fragments can be obtained using conventional recombinant and/or enzymatic techniques and can be screened for antigen binding in the same manner as intact antibodies.
  • binding refers to an association, which may be a stable association, between two molecules, for example, between a polypeptide and an antibody, due to, for example, electrostatic, hydrophobic, ionic and/or hydrogen-bond interactions under physiological conditions.
  • C R refers to a complementarity determining region (CDR) of an antibody or antibody fragment, which determine the binding character of an antibody or antibody fragment.
  • CDRs are present in a light chain variable region (CDRLl, CDRL2 and CDRL3) and three CDRs are present in a heavy chain variable region (CDRH1, CDRH2 and CDRH3).
  • CDRs contribute to the functional activity of an antibody molecule and are separated by amino acid sequences that comprise scaffolding or framework regions.
  • the CDR3 sequences, and particularly CDRH3 are the most diverse and therefore have the strongest contribution to antibody specificity.
  • CDRs There are at least two techniques for determining CDRs: (1) an approach based on cross-species sequence variability (i.e., Kabat et al., Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md. (1987), incorporated by reference in its entirety); and (2) an approach based on crystallographic studies of antigen-antibody complexes (Chothia et al., Nature, 342:877 (1989), incorporated by reference in its entirety).
  • cross-species sequence variability i.e., Kabat et al., Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md. (1987), incorporated by reference in its entirety
  • crystallographic studies of antigen-antibody complexes Chothia et al., Nature, 342:877 (1989), incorporated by reference in its entirety.
  • epitope means a protein determinant capable of specific binding to an antibody.
  • Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains. Certain epitopes can be defined by a particular sequence of amino acids to which an antibody is capable of binding.
  • extracellular epitope refers to an epitope that is located on the outside of a cell's plasma membrane. Exemplary extracellular epitopes of MRP9 are provided in Table 1 and Table 2.
  • the term “monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies that specifically bind to the same epitope, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts.
  • the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • telomere binding refers to the ability of an antibody to bind to a predetermined antigen or epitope.
  • an antibody specifically binds to its predetermined antigen or epitope with an affinity corresponding to a K D of about 10 "7 M or less, and binds to the predetermined antigen/binding partner with an affinity (as expressed by K D ) that is at least 10 fold less, at least 100 fold less or at least 1000 fold less than its affinity for binding to a non-specific and unrelated antigen/binding partner ⁇ e.g., BSA, casein).
  • Multidrug Resistance-Associated Protein 9 (MRP9) is also known as ATP-Binding
  • MRP9 is a member of the superfamily of ATP -binding transporters and a member of the MRP subfamily, which is generally involved in multi-drug resistance. Increased expression of MRP9 has been associated with breast cancer. As described herein, MRP9 is specifically expressed on the surface of GSCs and is therefore a useful target for GSC purification.
  • MRP9 is a well conserved protein among vertebrates.
  • the human MRP9 mRNA sequence is provided in Figure 3 and the human MRP9 protein sequence is provided in Figure 4 (with putative extracellular sequences capitalized and bold).
  • the mouse MRP9 mRNA sequence is provided in Figure 5 and the mouse MRP9 protein sequence is provided in Figure 6 (with putative extracellular sequences capitalized and bold).
  • Sequences for orthologous MRP9 proteins are known in the art and readily available via the NCBI database, including rat (NP_955409.1), zebrafish (XP_009310629.1), chimpanzee
  • the compositions and methods provided herein relate to antibodies (including antigen binding fragments thereof) that bind specifically to MRP9.
  • the MRP9 protein is human ⁇ e.g., a human MRP9 protein having an amino acid sequence of SEQ ID NO: 2), mouse ⁇ e.g., a mouse MRP9 protein having an amino acid sequence of SEQ ID NO: 4, or an orthologue thereof ⁇ e.g., a rat, zebrafish, chimpanzee, cow, monkey, dog, camel, bison, horse, goat, cat or sheep MRP9 protein).
  • the antibodies bind to a particular epitope of MRP9.
  • the epitope is an extracellular epitope.
  • the epitope is part of an extracellular domain of MRP9 ⁇ i.e. an extracellular epitope).
  • Exemplary extracellular domain sequences of human MRP9 are listed in Table 1, while exemplary mouse extracellular domain sequences of mouse MRP9 are listed in Table 2.
  • the antibody specifically binds to a polypeptide of SEQ ID NO: 6.
  • the antibody specifically binds to a polypeptide of SEQ ID NO: 7.
  • the antibody specifically binds to a polypeptide of SEQ ID NO: 8.
  • the antibody specifically binds to a polypeptide of SEQ ID NO: 9.
  • the antibody specifically binds to a polypeptide of SEQ ID NO: 10 . In some embodiments, the antibody specifically binds to a polypeptide of SEQ ID NO: 11 . In some embodiments, the antibody specifically binds to a polypeptide of SEQ ID NO: 12 . In some embodiments, the antibody specifically binds to a polypeptide of SEQ ID NO: 14 . In some embodiments, the antibody specifically binds to a polypeptide of SEQ ID NO: 15 . In some embodiments, the antibody specifically binds to a polypeptide of SEQ ID NO: 16 . In some embodiments, the antibody specifically binds to a polypeptide of SEQ ID NO: 17.
  • the antibody specifically binds to a polypeptide of SEQ ID NO: 18. In some embodiments, the antibody specifically binds to a polypeptide of SEQ ID NO: 19. In some embodiments, the antibody specifically binds to a polypeptide of SEQ ID NO: 20.
  • Table 1 Exemplary human MRP9 extracellular domain sequences.
  • the antibody does not specifically bind to a DDX4 protein from the same species as the MRP9 protein to which it binds (e.g., a human DDX4 protein, a mouse DDX4 protein).
  • DDX4 protein sequences are known in the art and readily available through the NCBI database, including human (NP 001136021.1) and mouse (NP 00139357.1), each of which is hereby incorporated by reference.
  • the antibody does not specifically bind to a MRP9 epitope having an amino acid sequence of APNPVDD.
  • the antibody is polyclonal. In some embodiments, the antibody is monoclonal. In some embodiments, the MRP9 antibody can be of any species. In some embodiments, the antibody is a mouse, rat, sheep, goat, camel, chicken, duck, hamster, guinea pig, dog, monkey, human or synthetic antibody or a combination thereof.
  • Polyclonal antibodies can be prepared by immunizing a suitable subject (e.g. a mouse, rat, sheep, goat, camel, chicken, duck, hamster, guinea pig, dog, monkey, etc.) with a polypeptide immunogen (e.g., a MRP9 protein (e.g., a protein of SEQ ID NO: 2 or 4) or a fragment thereof (e.g., a polypeptide comprising SEQ ID NO: 5-20).
  • a suitable subject e.g. a mouse, rat, sheep, goat, camel, chicken, duck, hamster, guinea pig, dog, monkey, etc.
  • a polypeptide immunogen e.g., a MRP9 protein (e.g., a protein of SEQ ID NO: 2 or 4) or a fragment thereof (e.g., a polypeptide comprising SEQ ID NO: 5-20).
  • the polypeptide immunogen comprises an extracellular epitope of MRP9 (
  • the polypeptide antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized polypeptide.
  • ELISA enzyme linked immunosorbent assay
  • the antibody directed against the antigen can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as protein A chromatography to obtain the IgG fraction.
  • antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies using standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975) Nature 256:495-497) (see also Brown et al. (1981) J.
  • an immortal cell line typically a myeloma
  • lymphocytes typically splenocytes
  • the culture supematants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds to the polypeptide antigen, preferably specifically.
  • a monoclonal antibody that binds to a target protein described herein can be obtained by screening a recombinant combinatorial immunoglobulin library ⁇ e.g., an antibody phage display library or an antibody yeast display library) with the appropriate polypeptide such as a MRP9 protein ⁇ e.g., a protein of SEQ ID NO: 2 or 4) or a fragment thereof ⁇ e.g., a polypeptide comprising SEQ ID NO: 5-20), to thereby isolate immunoglobulin library members that bind the polypeptide.
  • a recombinant combinatorial immunoglobulin library ⁇ e.g., an antibody phage display library or an antibody yeast display library
  • the appropriate polypeptide such as a MRP9 protein ⁇ e.g., a protein of SEQ ID NO: 2 or 4
  • a fragment thereof e.g., a polypeptide comprising SEQ ID NO: 5-20
  • recombinant antibodies specific for a target protein provided herein and/or an extracellular epitope of a target protein provided herein can be made using standard recombinant DNA techniques.
  • Such chimeric antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in US Pat No. 4,816,567; US Pat. No. 5,565,332; Better et al. (1988) Science 240: 1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci.
  • Human monoclonal antibodies specific for a target protein provided herein and/or an extracellular epitope of a target protein provided herein can be generated using transgenic or transchromosomal mice carrying parts of the human immune system rather than the mouse system.
  • transgenic or transchromosomal mice carrying parts of the human immune system rather than the mouse system.
  • “HuMAb mice” which contain a human immunoglobulin gene miniloci that encodes unrearranged human heavy ( ⁇ and ⁇ ) and ⁇ light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous ⁇ and ⁇ chain loci (Lonberg, N. et al. (1994) Nature 368(6474): 856 859).
  • mice exhibit reduced expression of mouse IgM or ⁇ , and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high affinity human IgGK monoclonal antibodies (Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N. (1994) Handbook of Experimental Pharmacology 113 :49 101; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. Vol. 13 : 65 93, and Harding, F. and Lonberg, N. (1995) Ann. N. Y Acad. Sci 764:536 546).
  • the preparation of HuMAb mice is described in Taylor, L. et al. (1992)
  • the antibodies provided herein are able to bind to MRP9 protein and/or a portion of MRP9 listed in Table 1 or Table 2 ⁇ e.g., an extracellular domain) with a dissociation constant of no greater than 10 "6 , 10 "7 , 10 "8 or 10 "9 M.
  • a dissociation constant of no greater than 10 "6 , 10 "7 , 10 "8 or 10 "9 M.
  • the antibodies provided herein do not bind to DDX4 with a dissociation constant of less than 10 "6 , 10 "7 , 10 "8 or 10 "9 M.
  • Standard assays to evaluate the binding ability of the antibodies are known in the art, including for example, ELISAs, Western blots and RIAs.
  • the binding kinetics ⁇ e.g., binding affinity) of the antibodies also can be assessed by standard assays known in the art, such as by Biacore analysis.
  • antibodies described herein is linked to a detectable label or a solid support.
  • detectable labels include fluorescent moieties, radioactive moieties, paramagnetic moieties, luminescent moieties and/or colorimetric moieties.
  • the antibodies described herein are linked to a fluorescent moiety. Examples of fluorescent moieties include, but are not limited to, Allophycocyanin,
  • Fluorescein Phycoerythrin, Peridinin-chlorophyll protein complex, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, EGFP, mPlum, mCherry, mOrange, mKO, EYFP, mCitrine, Venus, YPet, Emerald, Cerulean and CyPet.
  • Alexa Fluor 350 Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor
  • the methods provided herein include the step of purifying and/or detecting a population of GSCs, such as OSCs or SSCs ⁇ e.g., isolating GSCs from a sample and/or detecting GSCs in a sample).
  • the sample can be derived from any animal.
  • the sample is a human sample.
  • the sample is derived from a non-human animal.
  • the animal is a mammal.
  • the animal is a non-human mammal.
  • the mammal is a domesticated mammal ⁇ e.g., a cow, a pig, a horse, a donkey, a goat, a camel, a cat, a dog, a guinea pig, a rat, a mouse, a sheep, a zebu, a water buffalo, a yak, a llama, an alpaca, a ferret, a rabbit, a caribou, a reindeer).
  • the methods provided herein include the step of obtaining the sample from the animal.
  • the sample is a tissue sample. In some embodiments, the
  • GSCs are OSCs.
  • the OSCs are isolated from the ovarian tissue of the animal ⁇ i.e., from an ovarian tissue sample).
  • the GSCs are SSCs.
  • SSCs are isolated from testes tissue ⁇ i.e., from a testes tissue sample).
  • any technique known in the art and/or described herein can be used to purify and/or detect the GSCs based or MRP9 expression.
  • the GSCs are contacted with an anti-MRP9 antibody ⁇ e.g., an anti-MRP9 specific antibody described herein) under conditions such that the anti-MRP9 ant-MRP9 antibody forms a complex with MRP9 expressed on the surface of the GSCs.
  • the cells are washed ⁇ e.g., by centrifugation) such that unbound antibody is removed from the sample.
  • GSCs are then purified and/or detected by purifying or detecting cells to which the anti-MRP9 antibody has bound.
  • the anti-MRP9 antibody is either directly linked to a detectable label or is indirectly associated with a detectable label (e.g., through a secondary antibody or protein that specifically binds to the anti-MRP9 antibody) and the GSCs are purified and/or detected by targeting cells to which the detectable label is bound.
  • the detectable label is a fluorescent label and the GSC is purified and/or detected by fluorescent activated cell sorting (FACS) or fluorescence microscopy.
  • FACS fluorescent activated cell sorting
  • the detectable label is a paramagnetic particle and the GSC is purified and/or detected by magnetic activated cell sorting (MACS).
  • the anti-MRP9 antibody is either directly immobilized on a solid support or is indirectly immobilized with a solid support (e.g., through a secondary antibody or protein that specifically binds to the anti-MRP9 antibody) and the GSCs are purified by separating the cells bound to the solid support from other material present in the sample using adhesion based cell sorting.
  • the protein that specifically binds to the anti-MRP9 antibody is Protein A, Protein C and/or Protein G.
  • GSCs purified according to the methods described herein are then grown in culture.
  • Methods for growing GSCs in culture are described, for example, in Zou et al, Nat. Cell. Biol. 11 :631-636 (2009), Woods and Tilly, Nature Protocols 8:966- 988 (2013), White et al, Nature Medicine 18:413-421 (2012), Kanatsu-Shinohara et al, Biol. Reprod. 69:612-616 (2003) and Kanatsu-Shinohara et al, Biol. Reprod. 72:985-991 (2005), each of which is hereby incorporated by reference in its entirety.
  • Example 1 MRP9 is a marker for isolating oogonial stem cells
  • DDX4 antibody (Abeam 13840) commonly used in the purification of oogonial stem cells (OSCs) binds to DDX4 on live cells
  • OSCs oogonial stem cells
  • OSCs were grown in MEM-a GlutaMax (Invitrogen 32561) with 10% (v/v) FBS (Invitrogen 26140), 1 mM sodium pyruvate (Invitrogen 11360), 0.1 mM EAA (Invitrogen 11140), pen-strep-glutamine (Invitrogen 10378), N-2 Plus supplement (R&D Systems 212- GD-050) , 0.1 mM ⁇ -mercaptoethanol, 1000 units/mL LIF (Millipore ESGl 106), 10 ng/mL EGF (Invitrogen PHG0314), 1 ng/mL bFGF (Invitrogen 13256), and 40 ng/mL GD F
  • Protein content was determined by the BCA assay (Pierce). Proteins were run on SDS-PAGE under reducing conditions. The separated proteins were then electrophoretically transferred to a polyvinylidene difluoride membrane (Perkin- Elmer). Proteins of interest were revealed with specific antibodies: anti- DDX4 (Abeam 13840) and anti-a-tubulin, overnight at 4°C. The immunostaining was detected using horseradish peroxidase-conjugated anti-rabbit or anti-mouse immunoglobulin for 1 hour at room temperature. Bands were detected using ECL detection reagents (GE Healthcare).
  • siRNA knockdown of MRP9 was employed.
  • siMRP9# ⁇ D-060119-02, GE Healthcare, AACACCAUUCACGACAUA
  • siMRP9#2 D-060119-03, GE Healthcare
  • Proteins of interest were revealed with specific antibodies: anti-MRP9 (A viva OAAB9500), and anti-a-tubulin, overnight at 4°C.
  • the immunostaining was detected using horseradish peroxidase-conjugated anti-rabbit or anti-mouse immunoglobulin for 1 hour at room temperature. Bands were detected using ECL detection reagents (GE Healthcare).
  • Example 2 Isolating OSCs based on MRP9 expression
  • Ovarian tissue is obtained and minced into a slurry in a small volume of a collagenase/DNase I solution (Type IV Collagenase (Worthington LS004188) and DNase I (Roche 04536282001) in HBSS).
  • the slurry is collected and incubated in an orbital shaker at 37°C until tissue is digested into a cell suspension.
  • the cell suspension is filtered through a 70- ⁇ nylon mesh cell strainer and the filtrate collected in a 15-ml conical collection tube.
  • the volume of the filtrate is increased to 10 mL by washing the digestion tube with HbSS and filtering further into the collection tube.
  • the strained cell suspension is centrifuged at 300 x g at 4°C for 15 minutes.
  • the liquid is removed and the cell pellet resuspended in blocking buffer (2% bovine serum albumin and 2% normal goat serum in HBSS). About 90-95% of the resuspended cell solution are transferred to the primary sample tube, with the remaining cell solution transferred to separate tubes and used as controls (unstained control, anti-MRP9 antibody only control, secondary antibody only control, DAPI only control). Samples and controls are incubated on ice for 10-15 minutes. HBSS is added to all of the tubes and the anti-MRP9 antibody only control and Sample tubes and cells in these tubes are centrifuged at 300 x g at 4°C for 5 minutes.
  • blocking buffer 2% bovine serum albumin and 2% normal goat serum in HBSS.
  • the supernatant is removed from the anti- MRP9 antibody only control and Sample tubes and the cell pellets are resuspended in anti- MRP9 antibody solution (mouse monoclonal antibody specific for an MRP9 extracellular domain epitope diluted in blocking buffer).
  • anti- MRP9 antibody solution mouse monoclonal antibody specific for an MRP9 extracellular domain epitope diluted in blocking buffer.
  • the anti-MRP9 antibody only control and Sample tubes are incubated on ice for 10-15 minutes, at which time HBSS is added to the anti-MRP9 antibody only control, secondary antibody only control and Sample tubes, which are centrifuged at 300 x g at 4°C for 5 minutes.
  • the anti-MRP9 antibody only control cell pellet is resuspended in HBSS while the secondary antibody only control and sample cell pellets are resuspended in secondary antibody solution (APC labeled anti- mouse IgG antibody diluted in blocking buffer).
  • the secondary antibody only control and sample tubes are incubated on ice for 10-15 minutes.
  • HBSS is added to the secondary antibody only control and sample tubes and all tubes (controls and sample) are centrifuged at 300 x g at 4°C for 5 minutes. The supernatant is removed from all tubes.
  • the DAPI only control and sample cell pellets are resuspended in FACS buffer (0.5% FBS in HBSS) containing 1 ⁇ g/ml DAPI, while the remaining control cell pellets are resuspended in FACS buffer alone. Resuspended cells are filtered into FACS tubes. The control cells are used to calibrate the cell sorter and the MRP9 + and DAPT cells are purified from the sample by FACS.
  • the primary anti- MRP9 antibody could be directly conjugated to a fluorphore, rendering the secondary antibody unnecessary.
  • the primary antibody or the secondary antibody could be conjugated to a paramagnetic particle and the MRP9 positive cells purified by MACS instead of FACS.
  • MRP 9. but not DDX4, is expressed on the cell surface of OSCs
  • the cellular localization of DDX4 and MRP9 was determined using cultured OSCs.
  • Whole cell homogenates, cytosolic fractions and membrane fractions of cultured OSCs were isolated using gentle lysis and differential specification. Specifically, approximately 8 x 10 8 OSCs were raised in OCC culture to 80% confluency. Cells were scraped, washed twice in PBS and pelleted. Whole cells were homogenized and cytosolic versus membrane fractions were purified and prepared for SDS-page and immunoblotting using the Abeam Plasma Membrane Purification Protein Extraction Kit (ab65400).
  • the immunostaining was detected using horseradish peroxidase-conjugated anti -rabbit or anti-mouse immunoglobulin for 1 hour at room temperature. Bands were detected using ECL detection reagents (GE Healthcare). As seen in Figure 7, DDX4 is only present in the cytosolic fraction, while MRP9 is abundantly present in both the membrane and cytosolic fractions

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne des méthodes et des compositions pour la purification et la détection de cellules souches germinales (par exemple, des cellules souches ovogoniales) basées sur l'expression de MRP9.
EP16737697.9A 2015-01-13 2016-01-12 Purification de cellules souches germinales par ciblage de mrp9 Withdrawn EP3245287A4 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201562102820P 2015-01-13 2015-01-13
PCT/US2016/012964 WO2016115077A1 (fr) 2015-01-13 2016-01-12 Purification de cellules souches germinales par ciblage de mrp9

Publications (2)

Publication Number Publication Date
EP3245287A1 true EP3245287A1 (fr) 2017-11-22
EP3245287A4 EP3245287A4 (fr) 2018-06-20

Family

ID=56406270

Family Applications (1)

Application Number Title Priority Date Filing Date
EP16737697.9A Withdrawn EP3245287A4 (fr) 2015-01-13 2016-01-12 Purification de cellules souches germinales par ciblage de mrp9

Country Status (4)

Country Link
US (1) US20180002662A1 (fr)
EP (1) EP3245287A4 (fr)
AU (1) AU2016206945A1 (fr)
WO (1) WO2016115077A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
MX358243B (es) * 2014-09-16 2018-08-10 Ovascience Inc Anticuerpos anti-vasa y metodos de produccion y uso de los mismos.

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005105984A2 (fr) * 2004-04-12 2005-11-10 The Trustees Of The University Of Pennsylvania Conditions de culture et facteurs de croissance modifiant une determination de devenir, autorenouvellement et dilatation de cellules souches spermatogoniales de souris
US20100330043A1 (en) * 2006-10-23 2010-12-30 Cornell Research Foundation, Inc. Gpr125 as a marker for stem and progenitor cells and methods use thereof
US20080159999A1 (en) * 2006-10-23 2008-07-03 Konstantinos Stefanidis Compositions and methods for identifying, isolating and enriching germline-like stem cells from amniotic fluid
WO2009021151A1 (fr) * 2007-08-07 2009-02-12 Primegen Biotech Llc Isolement, caractérisation et propagation de cellules souches germinales

Also Published As

Publication number Publication date
EP3245287A4 (fr) 2018-06-20
US20180002662A1 (en) 2018-01-04
AU2016206945A1 (en) 2017-08-10
WO2016115077A1 (fr) 2016-07-21

Similar Documents

Publication Publication Date Title
KR102102111B1 (ko) Ｃｄｉｍ 결합 단백질 및 이의 용도
Sachen et al. Self-antigen recognition by follicular lymphoma B-cell receptors
Lipman et al. Monoclonal versus polyclonal antibodies: distinguishing characteristics, applications, and information resources
KR102661060B1 (ko) 항인간 IgG4 모노클로날 항체, 및 그 항체를 이용한 인간 IgG4 측정 시약
KR102427948B1 (ko) 다발성 골수종에서 m-단백질 반응의 임상 평가
US20170320966A1 (en) Humanized monoclonal antibody and uses thereof
WO2012147992A1 (fr) Anticorps spécifique anti-cellules ips/es et son utilisation
US20180002662A1 (en) Purification of germ stem cells by targeting mrp9
KR101971224B1 (ko) 면역학적 방법을 이용한 콜레스테롤 측정방법
WO2020204977A1 (fr) Anticorps anti-récepteur nmda et procédés d'utilisation
CN116375867A (zh) 抗gprc5d抗体及其应用
Lee et al. Identification of secreted and membrane-bound bat immunoglobulin using a Microchiropteran-specific mouse monoclonal antibody
US20170037366A1 (en) Enrichment and characterization of human corneal endothelial cells (hcenc) with novel monoclonal antibody
Albitar Monoclonal antibodies: methods and protocols
US11313864B2 (en) Method for determining anti-drug antibodies in a minipig sample
US11913951B2 (en) High throughput method for measuring the protease activity of complement C3 convertase
US20230314450A1 (en) METHOD FOR DETECTING A ß-SHEET AGGREGATE FORM OF A PROTEIN FORMING ß-SHEET AGGREGATES
US20120322083A1 (en) Antibody binding to abca1 polypeptide
Venkataraman et al. A toolbox of immunoprecipitation-grade monoclonal antibodies against human transcription factors
Cowan Development and Characterization of Novel Anti-bat Monoclonal Antibodies for Studying Bat Immunity
Dorschner et al. Listening to the Whispers in Neuroimmune Crosstalk: A Comprehensive Workflow to Investigate Neurotrophin Receptor p75NTR Under Endogenous, Low Abundance Conditions
Vergroesen et al. Pronounced (intrinsic) differences in N-glycosylation sites located in the variable domain of Igκ and Igλ light chains in ACPA-expressing B cells
KR101931617B1 (ko) 돼지의 이소글로보트리헥소실세라미드 신세타제(iGb3s)에 대한 항체 및 이의 용도
CN117700562A (zh) 靶向嘌呤霉素的兔单克隆抗体及其应用
CN116554336A (zh) 特异性结合Cas9的抗体或其抗原结合片段

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20170810

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

AX Request for extension of the european patent

Extension state: BA ME

DAV Request for validation of the european patent (deleted)
DAX Request for extension of the european patent (deleted)
A4 Supplementary search report drawn up and despatched

Effective date: 20180524

RIC1 Information provided on ipc code assigned before grant

Ipc: G01N 15/14 20060101ALI20180517BHEP

Ipc: C12N 5/075 20100101AFI20180517BHEP

Ipc: G01N 27/72 20060101ALI20180517BHEP

Ipc: G01N 33/532 20060101ALI20180517BHEP

Ipc: C12N 5/077 20100101ALI20180517BHEP

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20200801